- Email: [email protected]
Relative liver tumor promoting activity of some polychlorinated dibenzo-p-dioxin-, dibenzofuran- and biphenyl-congeners in female rats
Chemosphere, Voi.25, Nos.l-2, Printed in Great Britain
pp 169-172,
1992
0045-65135/92 $5.00 + 0.00 Pergamon Press Ltd.
RELATIVE LIVER TUMOR PROMOTING ACTIVITY OF SOME POLYCHLORINATED DIBENZO-p-DIOXIN-, DIBENZOFURAN- AND BIPHENYLCONGENERS IN FEMALE RATS
S. Flodstr6m and U.G. Ahlborg Institute of Environmental Medicine, Karolinska Institutet, Box 60208, S-104 01 Stockholm, Sweden
ABSTRA~ Some pentachlorinated dibenzo-p-dioxin-, dibenzofuran- and biphenyl-congeners and TCDD were studied for liver tumor promoting activity in a medium-term altered loci assay with nitrosamine initiated female SD rats. The congeners under study were administered by weekly subcutaneous injections for 20 weeks. Evaluation of GGT + or GST-P + altered foci development showed that all congeners studied acted as promoters of hepatocarcinogenesis. TCDD and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) were virtually equipotent as enhancers of loci development while 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) displayed approximately 10% of the activity of the dioxins. The PCB congener 2,3,3',4,4'pentachlorobiphenyl (PCB105) also enhanced foci development but was more than 1000 times less potent than TCDD. Analysis of the dioxin- and furan-eongeners by GC/MS-technique showed that the retention of PeCDD and PeCDF in liver tissue was approximately 7 and 20 times, respectively, as high as the retention of TCDD.
INTRODUCTION The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to be carcinogenic in several rodent species and acts as a highly potent promoter of hepatocarcinogenesis in female rats and skin tumors in mice (1). In recent risk assessments of environmentally occurring and toxic PCDD-, PCDF- and PCB-congeners, the use of Toxic Equivalency Factors CI'EF) have been suggested, relating the toxicities of individual congeners to the toxicities of TCDD (2,3). However, only a few of the PCDD, PCDF and PCB-congeners with dioxin-like activity have been studied for carcinogenic or tumor promoting activity, despite the fact that such effects have been crucial in the risk assessment of TCDD. In this study, we have investigated the relative liver tumor promoting activity of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7,8pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,3',4,4'pentachlorobiphenyl (PCB105) as measured by the enhancement of altered hepatic loci development. M~HODS In this study, the two-stage model of initiation-promotion with quantification of altered hepatic foci (AHF) development in female Sprague Dawley rats was used (4). Young rats (-120 g) were initiated by nitrosodiethylamine (30 mg/kg, i.p.) 24 hours after a 2/3-partial hepatectomy and allowed to recover for five weeks. At that time, groups of rats were allocated to various treatment groups receiving the PCDD/PCDF/PCB-congeners by weekly i.p. injections at three dose levels or 169
170
only the vehicle for 20 weeks. The doses of PCDD/PCDF/PCB administered in the various treatment groups are shown in Table 1. An initial loading dose (5 x maintenance dose) was applied in each treatment group to attain steady state tissue levels more rapidly (5). Altered loci were identified by enzymehistochemical staining for "/-ghtamyltranspeptidase (GGT) activity or by immunohistochemical staining for placental glutathione-S-transferase (GST-P). The concentration of PCDD/PCDF-congeners in liver tissue was measured mainly as reported by Lindstr6m and Rappe (6).
RESULTS The results from the stereological evaluation of altered hepatic foci development in the various treatment groups are shown in Table 1. In the dioxin/furan experiment, all PCDD/PCDF-treated groups except the PeCDF-Iow group, showed enhanced loci development indicating a tumor promoting effect. TABLE 1 TREATMENT GROUP
PER CENT FOCI TISSUE IN LIVER
FOCI/CM 3
EROD ACTIVrrVa
CONC. IN LIVER (ng/g)
CONTROL I b
0.21 (0.03) c
254 (31)
95 (11)
TCDD 44 n~kg/wk
0.76 (0.14)*
524 (54)*
1413 (157)*
0.84 (0.22)
TCDD 175 ng/k~wk
0.78 (0.17)
473 (81)*
3117 (260)*
3.17 (0.55)
TCDD 700 ng/k&twk
1.39 (0.39)
856 (138)*
3712 (241)*
10.06 (1.53)
PeCDD 88 ng/kg/wk
0.47 (0.13)
345 (76)
2102 (619)
5.19 (0.43)
PeCDD 350 ng/kg/wk
1.01 (0.33)
562 (82)*
3254 (377)*
37.0 (3.82)
PeCDD 1400 ng/kg/wk
2.06 (0.46)
1030 (118)*
5633 (327)*
142.8 (4.85)
PeCDF 160 ng/k~wk
0.32 (0.06)
306 (47)
575 (92)
PeCDF 640 ng/kg/wk
0.57 (0.11)
426 (99)*
1881 (239)*
208.4 (18.2)
PeCDF 2600 ng/kg/wk
0.83 (0.30)*
480/101)*
3093 (600)*
560.2 (42.9)
CONTROL 2d
2.3 (0.23)
2522 (259)
TCDD I ug.tkg/wk
23.9 (4.3)
5288 (569)
1997 (186)*
ND
PCB126 10/,g/kg/wk
12.7 (3.1)
7651 (588)
1805 (230)*
ND
PCB126 100/~g/kg/wk
52.6 (7.4)
1007 (77)*
ND
478 (60)
ND
ND
13 (1)
47.7 (10.6)
NO e
PCB126 1000 uj~/kK/wk PCB105 500 ujz/k~'wk
2.9 (0.37)
4910 (654)
PCB105 1500/~g/kg/wk
5.0 (0.95)
5368 (521)
554 (93)
ND
PCBI05 5000/,g/kg/wk
4.2 (0.93)
5319 0064)
1074 (84)*
ND
pmol resorufirdmin/mg microsomal or $9 protein Control in the PCDD/PCDF experiment; GGT+ foci Mean (SE), N=10 except for concentration in liver where N=4 Control in the TCDD/PCB experiment; GST-P ÷ foci N D = Not determined Significantly different from relevant control group (p< 0.05; Duncans multiple range test on log-transformed data)
171
Fig. 1A attempts to superimpose the dose response graphs for PeCDD and PeCDF with TCDD. PeCDD and TCDD showed similar dose-response behavior while multiplication of the PeCDF-doses with 0.1 resulted in a response curve similar to those of the dioxins. In Fig. 1A and 1B, the low dose of TCDD was omitted from the curve as that point appeared to be out of range. 2.5
2.5
0 TCDD
o TCDD
•
PeCDD
.
PeCDD ." 0114
.~
yJ
n
t.5
1.5
"~
1.0
0.5
0.0
•
2.0
2.0
1.0
o.~
0
500
tO00 Dose [ n g / k g / w k ]
1,500
0.0
., .... 0
, .... 5
, .... I0
i...llill, 15
ConcentraUon
20
25
In l i v e r [rtg/ll]
Figure 1. Percentage of liver occupied by GGT + .loci plotted against weekly dose (tl) and concentration in the liver of the respective congener (B). Dose and concentration data for PeCDD and PeCDF were transformed as indicated in the figures. The results of the PCDD/PCDF analyses in liver tissue showed that PeCDD and PeCDF accumulated in the liver to a much higher extent than TCDD (Table 1). At comparable levels of exposure, the hepatic concentrations of PeCDD and PeCDF were 7 and 20 times, respectively, as high as for TCDD. In Fig. 1B, the percentage of the liver occupied by GGT* loci in the treatment groups has been plotted against the hepatic concentrations of the dioxinsdfuran. However, the concentrations PeCDD and PeCDF were multiplied by an appropriate factor to superimpose the concentration-response curves with TCDD. A factor of 0.14 was assigned to PeCDD, while PeCDF required a factor of 0.007 to match the TCDD concentration-response curve. Hepatic cytochrome P450c-related monooxygenase activity (EROD) in the various treatment groups is given in Table 1. All dioxin/furan-treated groups displayed a statistically significant and dose related increase in EROD-activity, however the dioxins appear to be more potent inducers than PeCDF. Furthermore, liver weights, plasma transaminase activities and the incidence of histopathological liver lesions (focal necrosis and hypertrophy of hepatocytes) in the various treatment groups showed that the dioxins induced more severe effects than did PeCDF (data not shown). As shown by the preliminary data in Table 1, the PCB congeners studied in the TCDD/PCB experiment were inducers of EROD activity and enhanced the development of GST-P ÷ loci. PCB126 administered at a dosage level of 1000/Jg/kg/wk proved to be lethal for the animals after some weeks of treatment. Furthermore, all rats maintained on the 100/zg/kg/wk PCB126-regimen had numerous, visible liver noduls (~ 1-5 ram). Trough comparison with the TCDD treated group it was determined that PCB126 was about 10 times less potent than TCDD as a promoter. Similarly, PCB105 displayed a promoting potency more than 1000 times lower than TCDD.
172
The present study has shown that TCDD, PeCDD, PeCDF, PCB126 and PCB105 act as promoters of hepatocarcinogenesis in female rats as indicated by their enhancing effects on altered hepatic loci development. TCDD and PeCDF have been shown earlier to possess tumor promoting activity in rat liver (7,8,9) and to be hepatocarcinogens in long-term rodent experiments (1,10). However, results from studies whereby the promotional potencies of TCDD and PeCDF can be compared have not been available. As for PeCDD, PCB126 and PCB105, no results regarding liver tumor promoting activity have been published. The relative tumor promoting activity of TCDD, PCB126 and PCB105 found in this study support the Toxic Equivalency Factors (TEFs) for PCB126 and PCB105 suggested by Safe (2) based on toxicity endpoints other than carcinogenicity and promoting activity. In earlier evaluations (2,3,11) TEFs of 0.5 have been suggested for both PeCDD and PeCDF. The results from the present study would suggest the assignment of different TEFs for these congeners. Based on their relative tumor promoting activities, TEFs of 1 and 0.1 seem to be appropriate for PeCDD and PeCDF, respectively (Fig. 1A). The relative potency values for PeCDD and PeCDF as tumor promoters, based on their concentration in liver tissue at the termination of the study (Fig. 1B) were significantly lower than the TEFs based on the weekly delivered doses of the congeners, reflecting the higher retention of PeCDD and PeCDF in the liver as compared to TCDD.
ACKNOWLEDGEMEN'I~ Research was supported by grants from the Swedish Work Environment Fund (91/0236) and by the National Swedish Environmental Protection Board (5323091-8).
1. WHO/IPCS (1989) Polychlorinated dibenzo-p-dioxins and dibenzofurans. Environmental Health Criteria 88, WHO, Geneva. 2. Safe, S. (1990) CRC Crit. Rev. Toxicol. 21(1), 51-881 3. Ahlborg, U.G., H~ansson, H., Waern, F., and Hanberg, A. (1988) Health risk assessment of PCDDs and PCDFs. Mflj6rapport 1988:7. The Nordic Council, Copenhagen (in Swedish with English summary). 4. Pitot, H.C., Barsness, L., Goldsworthy, T., and Kitigawa, T. (1978) Nature 271, 456-457. 5. Flodstr6m, S. and Ahlborg, U.G. (1989) Chemosphere 19(1-6), 779-783 6. Lindstr6m, G. and Rappe, C. (1990) Chemosphere 20(7-9), 851-856. 7. Pitot, H.C., Goldsworthy, T., Campbell, H.A., and Poland, A. (1980) Cancer Res., 40, 36163620. 8. Flodstr6m, S., Bask, L., Kronevi, T., and Ahlborg, U.G. (1991) Fundam. Appl. Toxicol., 16, 375-391. 9. Nishizumi, M. and Masuda, Y. (1986) Cancer Lett., 33, 333-339. 10. Nishizumi, M. (1989) Fukuoka Acta Med. 80(5)~ 240-245. 11. Kutz, F.W., Barnes, D.G., Bretthauer, E.W., Bottimore D.P. and Greim, H. (1990). Toxicol. Environ. Chem., 26, 99-109.
pp 169-172,
1992
0045-65135/92 $5.00 + 0.00 Pergamon Press Ltd.
RELATIVE LIVER TUMOR PROMOTING ACTIVITY OF SOME POLYCHLORINATED DIBENZO-p-DIOXIN-, DIBENZOFURAN- AND BIPHENYLCONGENERS IN FEMALE RATS
S. Flodstr6m and U.G. Ahlborg Institute of Environmental Medicine, Karolinska Institutet, Box 60208, S-104 01 Stockholm, Sweden
ABSTRA~ Some pentachlorinated dibenzo-p-dioxin-, dibenzofuran- and biphenyl-congeners and TCDD were studied for liver tumor promoting activity in a medium-term altered loci assay with nitrosamine initiated female SD rats. The congeners under study were administered by weekly subcutaneous injections for 20 weeks. Evaluation of GGT + or GST-P + altered foci development showed that all congeners studied acted as promoters of hepatocarcinogenesis. TCDD and 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD) were virtually equipotent as enhancers of loci development while 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB126) displayed approximately 10% of the activity of the dioxins. The PCB congener 2,3,3',4,4'pentachlorobiphenyl (PCB105) also enhanced foci development but was more than 1000 times less potent than TCDD. Analysis of the dioxin- and furan-eongeners by GC/MS-technique showed that the retention of PeCDD and PeCDF in liver tissue was approximately 7 and 20 times, respectively, as high as the retention of TCDD.
INTRODUCTION The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to be carcinogenic in several rodent species and acts as a highly potent promoter of hepatocarcinogenesis in female rats and skin tumors in mice (1). In recent risk assessments of environmentally occurring and toxic PCDD-, PCDF- and PCB-congeners, the use of Toxic Equivalency Factors CI'EF) have been suggested, relating the toxicities of individual congeners to the toxicities of TCDD (2,3). However, only a few of the PCDD, PCDF and PCB-congeners with dioxin-like activity have been studied for carcinogenic or tumor promoting activity, despite the fact that such effects have been crucial in the risk assessment of TCDD. In this study, we have investigated the relative liver tumor promoting activity of TCDD, 1,2,3,7,8-pentachlorodibenzo-p-dioxin (PeCDD), 2,3,4,7,8pentachlorodibenzofuran (PeCDF), 3,3',4,4',5-pentachlorobiphenyl (PCB126) and 2,3,3',4,4'pentachlorobiphenyl (PCB105) as measured by the enhancement of altered hepatic loci development. M~HODS In this study, the two-stage model of initiation-promotion with quantification of altered hepatic foci (AHF) development in female Sprague Dawley rats was used (4). Young rats (-120 g) were initiated by nitrosodiethylamine (30 mg/kg, i.p.) 24 hours after a 2/3-partial hepatectomy and allowed to recover for five weeks. At that time, groups of rats were allocated to various treatment groups receiving the PCDD/PCDF/PCB-congeners by weekly i.p. injections at three dose levels or 169
170
only the vehicle for 20 weeks. The doses of PCDD/PCDF/PCB administered in the various treatment groups are shown in Table 1. An initial loading dose (5 x maintenance dose) was applied in each treatment group to attain steady state tissue levels more rapidly (5). Altered loci were identified by enzymehistochemical staining for "/-ghtamyltranspeptidase (GGT) activity or by immunohistochemical staining for placental glutathione-S-transferase (GST-P). The concentration of PCDD/PCDF-congeners in liver tissue was measured mainly as reported by Lindstr6m and Rappe (6).
RESULTS The results from the stereological evaluation of altered hepatic foci development in the various treatment groups are shown in Table 1. In the dioxin/furan experiment, all PCDD/PCDF-treated groups except the PeCDF-Iow group, showed enhanced loci development indicating a tumor promoting effect. TABLE 1 TREATMENT GROUP
PER CENT FOCI TISSUE IN LIVER
FOCI/CM 3
EROD ACTIVrrVa
CONC. IN LIVER (ng/g)
CONTROL I b
0.21 (0.03) c
254 (31)
95 (11)
TCDD 44 n~kg/wk
0.76 (0.14)*
524 (54)*
1413 (157)*
0.84 (0.22)
TCDD 175 ng/k~wk
0.78 (0.17)
473 (81)*
3117 (260)*
3.17 (0.55)
TCDD 700 ng/k&twk
1.39 (0.39)
856 (138)*
3712 (241)*
10.06 (1.53)
PeCDD 88 ng/kg/wk
0.47 (0.13)
345 (76)
2102 (619)
5.19 (0.43)
PeCDD 350 ng/kg/wk
1.01 (0.33)
562 (82)*
3254 (377)*
37.0 (3.82)
PeCDD 1400 ng/kg/wk
2.06 (0.46)
1030 (118)*
5633 (327)*
142.8 (4.85)
PeCDF 160 ng/k~wk
0.32 (0.06)
306 (47)
575 (92)
PeCDF 640 ng/kg/wk
0.57 (0.11)
426 (99)*
1881 (239)*
208.4 (18.2)
PeCDF 2600 ng/kg/wk
0.83 (0.30)*
480/101)*
3093 (600)*
560.2 (42.9)
CONTROL 2d
2.3 (0.23)
2522 (259)
TCDD I ug.tkg/wk
23.9 (4.3)
5288 (569)
1997 (186)*
ND
PCB126 10/,g/kg/wk
12.7 (3.1)
7651 (588)
1805 (230)*
ND
PCB126 100/~g/kg/wk
52.6 (7.4)
1007 (77)*
ND
478 (60)
ND
ND
13 (1)
47.7 (10.6)
NO e
PCB126 1000 uj~/kK/wk PCB105 500 ujz/k~'wk
2.9 (0.37)
4910 (654)
PCB105 1500/~g/kg/wk
5.0 (0.95)
5368 (521)
554 (93)
ND
PCBI05 5000/,g/kg/wk
4.2 (0.93)
5319 0064)
1074 (84)*
ND
pmol resorufirdmin/mg microsomal or $9 protein Control in the PCDD/PCDF experiment; GGT+ foci Mean (SE), N=10 except for concentration in liver where N=4 Control in the TCDD/PCB experiment; GST-P ÷ foci N D = Not determined Significantly different from relevant control group (p< 0.05; Duncans multiple range test on log-transformed data)
171
Fig. 1A attempts to superimpose the dose response graphs for PeCDD and PeCDF with TCDD. PeCDD and TCDD showed similar dose-response behavior while multiplication of the PeCDF-doses with 0.1 resulted in a response curve similar to those of the dioxins. In Fig. 1A and 1B, the low dose of TCDD was omitted from the curve as that point appeared to be out of range. 2.5
2.5
0 TCDD
o TCDD
•
PeCDD
.
PeCDD ." 0114
.~
yJ
n
t.5
1.5
"~
1.0
0.5
0.0
•
2.0
2.0
1.0
o.~
0
500
tO00 Dose [ n g / k g / w k ]
1,500
0.0
., .... 0
, .... 5
, .... I0
i...llill, 15
ConcentraUon
20
25
In l i v e r [rtg/ll]
Figure 1. Percentage of liver occupied by GGT + .loci plotted against weekly dose (tl) and concentration in the liver of the respective congener (B). Dose and concentration data for PeCDD and PeCDF were transformed as indicated in the figures. The results of the PCDD/PCDF analyses in liver tissue showed that PeCDD and PeCDF accumulated in the liver to a much higher extent than TCDD (Table 1). At comparable levels of exposure, the hepatic concentrations of PeCDD and PeCDF were 7 and 20 times, respectively, as high as for TCDD. In Fig. 1B, the percentage of the liver occupied by GGT* loci in the treatment groups has been plotted against the hepatic concentrations of the dioxinsdfuran. However, the concentrations PeCDD and PeCDF were multiplied by an appropriate factor to superimpose the concentration-response curves with TCDD. A factor of 0.14 was assigned to PeCDD, while PeCDF required a factor of 0.007 to match the TCDD concentration-response curve. Hepatic cytochrome P450c-related monooxygenase activity (EROD) in the various treatment groups is given in Table 1. All dioxin/furan-treated groups displayed a statistically significant and dose related increase in EROD-activity, however the dioxins appear to be more potent inducers than PeCDF. Furthermore, liver weights, plasma transaminase activities and the incidence of histopathological liver lesions (focal necrosis and hypertrophy of hepatocytes) in the various treatment groups showed that the dioxins induced more severe effects than did PeCDF (data not shown). As shown by the preliminary data in Table 1, the PCB congeners studied in the TCDD/PCB experiment were inducers of EROD activity and enhanced the development of GST-P ÷ loci. PCB126 administered at a dosage level of 1000/Jg/kg/wk proved to be lethal for the animals after some weeks of treatment. Furthermore, all rats maintained on the 100/zg/kg/wk PCB126-regimen had numerous, visible liver noduls (~ 1-5 ram). Trough comparison with the TCDD treated group it was determined that PCB126 was about 10 times less potent than TCDD as a promoter. Similarly, PCB105 displayed a promoting potency more than 1000 times lower than TCDD.
172
The present study has shown that TCDD, PeCDD, PeCDF, PCB126 and PCB105 act as promoters of hepatocarcinogenesis in female rats as indicated by their enhancing effects on altered hepatic loci development. TCDD and PeCDF have been shown earlier to possess tumor promoting activity in rat liver (7,8,9) and to be hepatocarcinogens in long-term rodent experiments (1,10). However, results from studies whereby the promotional potencies of TCDD and PeCDF can be compared have not been available. As for PeCDD, PCB126 and PCB105, no results regarding liver tumor promoting activity have been published. The relative tumor promoting activity of TCDD, PCB126 and PCB105 found in this study support the Toxic Equivalency Factors (TEFs) for PCB126 and PCB105 suggested by Safe (2) based on toxicity endpoints other than carcinogenicity and promoting activity. In earlier evaluations (2,3,11) TEFs of 0.5 have been suggested for both PeCDD and PeCDF. The results from the present study would suggest the assignment of different TEFs for these congeners. Based on their relative tumor promoting activities, TEFs of 1 and 0.1 seem to be appropriate for PeCDD and PeCDF, respectively (Fig. 1A). The relative potency values for PeCDD and PeCDF as tumor promoters, based on their concentration in liver tissue at the termination of the study (Fig. 1B) were significantly lower than the TEFs based on the weekly delivered doses of the congeners, reflecting the higher retention of PeCDD and PeCDF in the liver as compared to TCDD.
ACKNOWLEDGEMEN'I~ Research was supported by grants from the Swedish Work Environment Fund (91/0236) and by the National Swedish Environmental Protection Board (5323091-8).
1. WHO/IPCS (1989) Polychlorinated dibenzo-p-dioxins and dibenzofurans. Environmental Health Criteria 88, WHO, Geneva. 2. Safe, S. (1990) CRC Crit. Rev. Toxicol. 21(1), 51-881 3. Ahlborg, U.G., H~ansson, H., Waern, F., and Hanberg, A. (1988) Health risk assessment of PCDDs and PCDFs. Mflj6rapport 1988:7. The Nordic Council, Copenhagen (in Swedish with English summary). 4. Pitot, H.C., Barsness, L., Goldsworthy, T., and Kitigawa, T. (1978) Nature 271, 456-457. 5. Flodstr6m, S. and Ahlborg, U.G. (1989) Chemosphere 19(1-6), 779-783 6. Lindstr6m, G. and Rappe, C. (1990) Chemosphere 20(7-9), 851-856. 7. Pitot, H.C., Goldsworthy, T., Campbell, H.A., and Poland, A. (1980) Cancer Res., 40, 36163620. 8. Flodstr6m, S., Bask, L., Kronevi, T., and Ahlborg, U.G. (1991) Fundam. Appl. Toxicol., 16, 375-391. 9. Nishizumi, M. and Masuda, Y. (1986) Cancer Lett., 33, 333-339. 10. Nishizumi, M. (1989) Fukuoka Acta Med. 80(5)~ 240-245. 11. Kutz, F.W., Barnes, D.G., Bretthauer, E.W., Bottimore D.P. and Greim, H. (1990). Toxicol. Environ. Chem., 26, 99-109.